1. Field of the Invention
The present invention provides avirulent Salmonella variants and various uses thereof, particularly in the production of Salmonella-specific lytic bacteriophages, pharmaceutical compositions, and feed additives.
2. Description of the Related Art
Currently over 2,000 Salmonella strains are generally classified into host-specific serotypes, and non-host-specific serotypes pathogenic for both animals and humans. Representative among fowl-adapted pathogens are Salmonella gallinarum (SG) and Salmonella pullorum (SP) which are known to cause fowl typhoid and pullorum disease, respectively. These Salmonella-caused fowl diseases occur at low frequency in advanced countries, but have inflicted tremendous economic damage on the poultry farming in developing countries.
Salmonella gallinarum strains have serologically the same somatic antigen (O-antigen) structures and are classified as being non-motile because they have no flagella. When entering into a host animal via contaminated feed or a contaminated environment, Salmonella pass through the gastrointestinal tract, and invade intestinal epithelial cells by interaction with Peyer's patch M (microfold) cells and penetrate into the intestinal membrane. Salmonella are transported by the M cells to macrophages in adjacent intestinal membranes, and then Salmonella infection develops into a systemic disease.
The type III secretion system (TTSS) is a protein appendage found in Gram-negative bacteria, which consists of a needle-like protein complex structure through which virulence effector proteins pass from the bacterial cytoplasm directly into the host cytoplasm (Mota L J et al., Ann Med. (2005); 37(4):234-249). The type III secretion system is essential for the delivery of the pathogenicity of Salmonella (Schlumberger M C et al., Curr Opin Microbiol. (2006); 9(1):46-54). Wild-type Salmonella take advantage of TTSS when adhering to and invading host cells, and then survives during the phagocytosis of macrophages and circulates throughout the body via the bloodstream, causing a systemic infection. Hence, Salmonella infection cannot proceed without the normal operation of TTSS. Salmonella pathogenicity island-1 (hereinafter referred to as “SPI-1”) is a discrete region of the Salmonella chromosome encoding the type III secretion system and virulent effector proteins which are necessary for invasion into intestinal epithelial cells in the early stage of infection (Kimbrough T G et al., Microbes Infect, (2002); 4(1):75-82). Salmonella pathogenicity island-2 (hereinafter referred to as “SPI-2”) is also a discrete region of the Salmonella chromosome encoding the type III secretion system and effector proteins which involved in survival and proliferation during phagocytosis by macrophages in intestinal immune organs or immune organs such as the spleen and the liver after translocation across epithelial cells (Waterman S R et al., Cell Microbiol, (2003); 5(8):501˜511, Abrahams G L, Cell Microbiol, (2006); 8(5):728-737). Genes within SPI-1 and SPI-2 and their functions are summarized in Table 1, below.
TABLE 1GeneCharacteristicsSPI-1avrAputative inner membrane proteinsprBtranscriptional regulatorhilCbacterial regulatory helix-turn-helixproteins, araC familyorgAputative flagellar biosynthesis/typeIII secretory pathway proteinprgKcell invasion protein; lipoprotein,may link inner and outer membranesprgJIHcell invasion proteinhilDregulatory helix-turn-helix proteins,araC familyhilAinvasion genes transcription activatoriagBcell invasion proteinsptPprotein tyrosine phosphatesicPchaperone, related to virulenceiacPputative acyl carrier proteinsipADCBcell invasion proteinsicAsurface presentation of antigens;secretory proteinsspaSRQPOsurface presentation of antigens;secretory proteinsinvJICBsurface presentation of antigens;secretory proteinsinvAEGFHinvasion proteinSPI-2ssaUTSRQPONSecretion system apparatusVMLKJIHGsseGFSecretion system effectorsscBSecretion system chaperonesseEDCSecretion system effectorsscASecretion system chaperonesseBASecretion system effectorssaESecretion system effectorssaDCBSecretion system apparatusssrASecretion system regulator:SensorcomponentssrBSecretion system regulator:transcriptional activator, homologouswith degU/uvrY/bvgA
In addition to these type III secretion systems, fimbriae gene (faeHI) (Edwards R A et al., PNAS (2000); 97(3):1258-1262) and the virulent factor (spvRABCD operon) present in virulent plasmids of Salmonella are implicated in the virulence of Salmonella (Gulig P A et al., Mol Microbiol (1993); 7(6):825-830).
Salmonella-caused fowl diseases are difficult to control because they are transmitted in various ways including egg transmission, and feed or environmental infection, and show high recurrence rates even after post-infectious treatment with antibiotics. Therefore, it is importance of preventing the onset of disease by using a vaccine as well as sanitizing breeding farms and feed. In the poultry industry, a lot of effort has been poured into the use of live vaccines (attenuated Salmonella gallinarum strains—SG9S, SG9R) and dead vaccines (gel vaccines, oil vaccines, etc.) to prevent the onset of fowl typhoid. However, the effects of the vaccine vary with the concentration of the vaccine used, the condition of the fowl vaccinated, and the environment of chicken houses. And, the efficacy of these vaccines is reported to be significantly lower than that of the vaccines for other diseases. Treatment with antibiotics, although reducing the lesion, converts infected fowls into chronic carriers (See: Incidence and Prevention of Hen Salmonellosis, the National Veterinary Research & Quarantine Service, Korea).
Therefore, new Salmonella-controlling approaches that are better than conventional vaccines or antibiotics are being demanded. Many scientists have recently paid attention to bacteriophages, which infect and lyse bacteria specifically and are safe to humans, as a potent alternative to antibiotics. There are many reports concerning the use of bacteriophages being used in the prevention or therapy of Salmonella diseases (Atterbury R J et al., Appl Environ Microbiol, (2007); 73(14):4543-4549) and as disinfectants or detergents to prevent the putrefaction of foods (PCT 1998-08944, PCT 1995-31562, EP 1990-202169, PCT 1990-03122), and concerning phage display techniques for diagnosis (Ripp S et al., J Appl Microbiol, (2006); 100(3):488-499), Salmonella vaccines prepared by deleting or modifying one or two genes within SPI-2 gene cluster have recently been disclosed (U.S. Pat. No. 6,923,957, U.S. Pat. No. 7,211,264, U.S. Pat. No. 7,887,816).
For industrial use, bacteriophages are produced by separating the phage progenies from the host cells lysed during the proliferation of bacteriphages which have been inoculated into the host cells cultured on a mass scale. As for bacteriophages specific for pathogenic bacteria, however, their lysates may contain the pathogenic host cells being not removed, and/or virulent materials such as pathogenic proteins of the host. This likelihood acts as a great risk factor to the safety of bacteriophages produced on the basis of pathogenic host cells.
Many bacteria have lysogenic phages on their chromosomes; however, most of the phages are cryptic and cannot produce progeny because of the accumulation of many mutations as ancestral remnants. Lysogenic phages, although inactive, may help the survival capacity of Salmonella upon host infection because they contain the genes necessary for lytic and lysogenic growth and some of the genes encode pathogenic factors. However, these genes are likely to undergo homologous recombination with the viral genome of other similar phages which newly infect animals, thus producing genetically modified phages. As for the typical Salmonella typhimurium, it has fels-1, fels-2, gifsy-1, and gifsy-2 prophages and two cryptic phages. In contrast, Salmonella gallinarum could be used as a phage-producing host since Salmonella gallinarum have neither prophages nor cryptic phages, and then are not genetically modified by recombination. (Edwards R A et al, Trends Microbiol, (2002); 10(2):94-99).
For the purpose of minimizing toxic remnants during progeny production and phage's opportunity for mutation, the present inventors designed the idea that the virulence gene clusters of Salmonella gallinarum could be inactivated for producing bacteriophages. There have no precedent cases wherein avirulent bacteria, which had been converted from virulent bacteria by inactivating a virulence gene cluster, were used as a bacteriophage host cell.
In addition to the production of bacteriophages, the Salmonella deprived of virulence by inactivating virulence gene clusters are themselves used for developing attenuated live vaccines for controlling Salmonella or applied to the bioindustry, guaranteeing significant added values.
In the present invention, avirulent Salmonella gallinarum variants obtained by inactivating at least one of the main Salmonella virulence gene clusters (SPI-1, SPI-2, spvRABCD and faeHI operons) are used as a bacteriophage-producing host cell and applied to various uses.